The hair-cell tip link is an essential component of the mechanotransduction apparatus in the inner ear. During the past four years my research has focused on the structural biology of hair-cell tip links. I plan to continue this line of research,as my long-term career goal is to lead a multidisciplinary research group that studies the molecular mechanisms of hair-cell mechanotransduction. The research work I did during my postdoctoral training answered some key questions about the elasticity of tip-link fragments, and how the tips of cadherin-23 and protocadheirn-15 interact with each other. However, this work also opened the door to explore the biophysics of the entire tip link, and produced computational predictions about the strength and elasticity of tip links tha need to be verified experimentally. My short-term goals are to determine the structure of the whole tip link, to measure the strength of the tip-link bond experimentally, and to establish how mechanical force is transmitted through the tip link and to the hair-cell transduction channel. To achieve my short term goals, and during the K99 mentored phase, I will learn how to use the Pichia pastoris system for expression of long extracellular tip link fragments involving multiple EC repeats. The Gaudet lab has the expertise and tools for large-scale expression of proteins in this system In parallel I will learn how to use the optical tweezers setup at the Corey lab. This involves learning how to use and align lasers, how to design and synthesize linkers between molecules and functionalized, micrometer-size polystyrene beads, as well as performing the actual experiments. Training will be received in two laboratories with an outstanding and exceptionally friendly group of researchers. I will directly benefit from interactions with experts in eukaryotic protein expression (Gaudet laboratory) and in the use of optical tweezers (Corey laboratory). I will also receive advice from Dr. Wesley Wong, member of my advisory committee and an expert in single-molecule force spectroscopy. All members of these laboratories will provide the perfect and unique interdisciplinary environment required for my training. In addition, I will have access to outstanding facilities at both the Corey and Gaudet laboratories. I will be using state-of-the-art equipment required for single-molecule force spectroscopy experiments, eukaryotic protein production, and X-ray crystallography. After completing the mentored phase, I will have enough expertise in a broad repertoire of computational and experimental techniques to start an independent career investigating the molecular mechanisms underlying the function of the entire tip link as outlined below. (Aim 1) Optical tweezers will be use to probe the strength of a dimeric bond formed by the tips of cadherin-23 and protocadherin-15, and of an expected tetrameric bond formed by longer domains of these molecules. The experiments may not only reveal unbinding forces, but also intermediate states and, along with simulations, molecular mechanisms associated with formation and rupture of the bond. The rupture force of the tip link bond will provide clues about the consequences of loud sound stimuli on hair cell function. (Aim 2) A structural model for the entire extracellular tip link will be obtained, to characterize heteregeous EC repeats, establish their elasticity, and determine whether interdigitation involving multiples EC repeats is feasible. Results will conclusively determine whether the tip link can be the hair-cell gating spring and provide a structural framework to interpret multiple deafness mutations. (Aim 3) The mechanical response of protocadherin-15's transmembrane domain embedded in a lipid bilayer, along with a membrane-gated channel, will be simulated to test whether force is conveyed directly, or indirectly through the lipids, to hair-cell transduction channels. Results wil provide in silico and in vitro platforms to test models of MS channel activation and clarify the controversial role of membrane tension in inner-ear mechanotransduction.